Parkinson’s disease protein DJ-1 regulates ATP synthase protein components to increase neuronal process outgrowth

a Coomassie staining of the protein bands present in submitochondrial vesicles (SMVs) is shown in the left panel. The right panel shows an immunoblot using an anti-DJ-1 antibody against solubilized protein from SMVs. The blot confirms that DJ-1 is present in the SMV subcellular fraction. b WT and mutant DJ-1 immunolocalize with ATP synthase β-subunit. Extracts from HEK293 cells expressing flag-tagged WT or mutant DJ-1 were prepared for coimmunoprecipitation analysis with anti-FLAG antibody. Proteins were revealed with the indicated antibodies in western blots. Note that endogenous ATP synthase β-subunit is copurified with WT DJ-1 and also with mutant DJ-1. c Purified WT and mutant DJ-1 immunolocalize with ATP synthase β-subunit in vitro. Purified flag-ATP synthase β-subunit protein was incubated with purified DJ-1. The mixture was immuno-precipitated with anti-DJ-1 antibody. The protein interaction was revealed by western blotting with the indicated antibodies. d The ability of SMVs to sequester H+ is improved by recombinant WT DJ-1 protein. Shown are ACMA (H+ indicator) fluorescence intensity changes induced in response to ATP in the presence of rat SMVs. A decrease in indicator intensity represents sequestration of H+ into the lumen of SMVs. Data were acquired every 30？s for 5？min in the presence of BSA control protein or recombinant protein (all proteins were added at 10？μg/100？μl final). For WT DJ-1 (n？=？6 samples), M3 DJ-1 (n？=？8) or M5 DJ-1 (n？=？8). WT DJ-1 protein maximizes the ability of SMVs to sequester H+ whereas mutant DJ-1 proteins have no effect. **p？<？0.01, *p？<？0.05, recombinant DJ-1 protein versus BSA control (n？=？6). Paired, two-tailed t-test. Error bars represent mean？±？SEM. e The ability of SMVs to sequester H+ is compromised in DJ-1？/？ SMVs. WT (n？=？7) and DJ-1？/？ (n？=？6). **p？<？0.01, *p？<？0.05, Error bars represent mean？±？SE